Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice

Myelin, an extension of the oligodendrocyte plasma membrane, wraps around axons to facilitate nerve conduction. Myelination is compromised in ATR-X intellectual disability syndrome patients, but the causes are unknown. We show that loss of ATRX leads to myelination deficits in male mice that are partially rectified upon systemic thyroxine administration. Targeted ATRX inactivation in either neurons or oligodendrocyte progenitor cells (OPCs) reveals OPC-intrinsic effects on myelination. OPCs lacking ATRX fail to differentiate along the oligodendrocyte lineage and acquire a more plastic state that favors astrocytic differentiation in vitro and in vivo. ATRX chromatin occupancy in OPCs greatly overlaps with that of the chromatin remodelers CHD7 and CHD8 as well as H3K27Ac, a mark of active enhancers. Overall, our data indicate that ATRX regulates the onset of myelination systemically via thyroxine, and by promoting OPC differentiation and suppressing astrogliogenesis. These functions of ATRX identified in mice could explain white matter pathogenesis observed in ATR-X syndrome patients.

Myelin is an extension of the oligodendrocyte (OL) plasma membrane that wraps itself around axons to increase conduction velocity, enabling efficient propagation of action potentials.A series of events are necessary to produce myelin, including proliferation and migration of oligodendrocyte precursor cells (OPCs), recognition of recipient axons, membrane expansion, wrapping of axons and the appropriate production of membrane components and myelin proteins for compaction 1 .
In the developing neocortex, the generation of neurons from neural progenitor cells eventually shifts towards production of astrocytes and then OLs 2 .The majority of primitive OPCs are generated postnatally from neural stem cells (NSCs) and intermediate glial progenitor cells (iGCs) through the expression of the oligodendrocyte basic-helix-loop-helix transcription factors Olig1 and Olig2 3 .Olig2 is expressed in gliogenic progenitors where it acts as a repressor of neuronal differentiation.Postnatally, Olig2 has been reported to support OL differentiation from OPCs and to suppress astrocytic lineage in these cells [4][5][6][7] .Downstream of Olig2, the SRY-box transcription factor 10 (Sox10) participates in a positive feedback loop to establish the OPC lineage [8][9][10] .Notably, Sox10 activates several OPC-critical genes, including Chondroitin sulfate proteoglycan (Cspg4; protein: neural glial antigen 2 (NG2)) and Platelet-derived growth factor receptor α (Pdgfrα) [11][12][13] .NG2 is a surface type I transmembrane core protein involved in cell migration and proliferation 14 , whereas PDGFRα promotes OPC proliferation, migration and survival 15,16 .Further commitment to the OL lineage starts with the generation of immature oligodendrocytes (iOLs) which, after cell cycle exit, express myelin regulatory factor (MRF) 17 .As iOLs mature and contact axons, they become highly branched and upregulate several myelin sheath proteins in order to encompass the axon 1,18 .The expression of myelin proteins is complex and regulated by several different transcription factors including the aforementioned Olig1 19 , Sox10 19,20 and MRF 17 but also Sp1 21 , Nkx2.2 22 and the ligand-bound thyroid hormone receptor [23][24][25] .
Myelination has been studied extensively in the context of demyelinating diseases like multiple sclerosis and leukodystrophies 26 .However, myelin deficits are also frequently observed in neurodevelopmental cognitive disorders like autism and schizophrenia and can contribute to intellectual disability [27][28][29] .This is exemplified by the ATR-X syndrome, an intellectual disability disorder caused by abnormalities in the ATRX chromatin remodeling protein 30 .Affected males often exhibit cognitive dysfunction, seizures and autistic features, among other developmental defects [31][32][33][34][35] .ATR-X syndrome patients also display abnormal myelination with a prevalence of 44% 33,34 making white matter abnormalities a supporting feature of the diagnosis.
ATRX belongs to the Switch/Sucrose non-fermenting (Swi/Snf) family of chromatin remodelers 36 , and can regulate gene expression through several mechanisms.In the newborn cortex, ATRX represses expression of imprinted genes by fostering long-range chromatin interactions mediated by CCCTC-binding factor (CTCF) and cohesin 37,38 .In embryonic stem cells, it promotes maintenance of the repressive H3K27me3 histone mark at polycomb target genes 39 .On the other hand, ATRX can promote gene transcription through the incorporation of histone H3.3 and facilitating RNA PolII elongation through G-rich sequences 40,41 .In testes, ATRX regulates imprinted genes 42 associates with the androgen receptor and facilitates transcription of its target genes 43 .ATRX also promotes the expression of αglobin by negatively regulating the histone variant macroH2A 44 .
The molecular and cellular basis of white matter abnormalities associated with ATRX dysfunction has not yet been investigated.Here, we report that deletion of Atrx in the central nervous system causes hypomyelination.While myelin levels and OL numbers were partially recovered upon administration of the thyroid hormone thyroxine (T4), this treatment failed to replenish OPCs, indicating that ATRX is required to maintain these progenitors independently of circulating T4 levels.Indeed, targeted inactivation of Atrx in Sox10-expressing OPCs resulted in hypomyelination, revealing cell autonomous functions of ATRX in OPCs.We show that ATRX occupies and maintains Olig2 gene expression.In the absence of ATRX, OPCs tend to revert to a more malleable state, and gain the ability to differentiate into astrocyte-like cells.We conclude that ATRX deficiency affects the developmental trajectory of glial precursors, which could be the underlying cause of defective myelination in the associated ATR-X syndrome.

Decreased myelination in the Atrx FoxG1Cre mouse forebrain
We previously reported that postnatal health and longevity are adversely affected in mice with ATRX deficiency in the forebrain, anterior pituitary and liver (Atrx FoxG1cre mice) 45,46 .These mice have a reduced lifespan, growth and hormone abnormalities, including low circulating levels of IGF-1 and T4 45,46 and exhibit aberrant gene expression in the liver 45 .To identify potential forebrain related differences in transcription between control and Atrx FoxG1Cre mice, we performed a microarray analysis of 3 littermate-matched pairs of P17 control and Atrx FoxG1Cre mice.Microarray analysis revealed a significant downregulation of genes involved in myelination and oligodendrocyte morphology (Fig. 1a, b, p < 0.05).Moreover, downregulated genes are highly expressed in mouse oligodendrocytes according to the Allan Brain Atlas single cell RNA gene sets (Fig. 1c).Given previous reports of white matter abnormalities in ATR-X syndrome patients 33,34 , we further explored whether the mice exhibit a similar phenotype.We examined the level of well-established myelin markers by immunofluorescence staining of brain cryosections.The results showed decreased level of myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) in the corpus callosum and adjacent cortex in Atrx FoxG1Cre compared to control mice at P20 (Fig. 1d).Western blot analysis of myelin associated glycoprotein (MAG), MOG and MBP showed that myelin protein expression is significantly decreased in Atrx FoxG1Cre forebrain compared to littermate matched controls (n = 4 each genotype; Student's T-test, MAG p = 0.006, MOG p = 0.005, MBP p = 0.009) (Fig. 1e, f).These data strongly suggest that the Atrx FoxG1Cre CNS is hypomyelinated.
Postnatal thyroxine treatment partially restores myelination deficits in Atrx FoxG1Cre mice Circulating levels of the thyroid hormone T4 in Atrx FoxG1Cre mice are less than half that of controls, most likely stemming from the deletion of Atrx in the anterior pituitary in these mice 45,46 .The thyroid hormone receptor is a transcription factor that requires its ligand, triiodothyronine (T3), to promote OL differentiation and the expression of several myelin genes [47][48][49][50][51][52] .We therefore tested whether postnatal administration of the prohormone T4 might rescue hypomyelination in Atrx FoxG1Cre mice.We performed daily injections of 0.1 mg/kg L-thyroxine (T4) or PBS from P0 to P14, as outlined in Fig. 2a, a dose previously shown to successfully elevate circulating T4 to control levels in Atrx FoxG1Cre mice 45 .
We first quantified the number of OLs after T4 treatment by immunofluorescence staining of P14 brain cryosections, using an antibody specific for the pan-OL marker Olig2.We observed a significant reduction of the proportion of Olig2 + cells (representing all OPCs and OLs) in the Atrx FoxG1Cre corpus callosum (27.6% of total DAPI + cells) compared to controls (44.9% of total DAPI + cells) and that this was partially recovered in Atrx FoxG1Cre mice treated with T4 (32.3%) (n = 3 for each genotype; p = 0.0013, one-way ANOVA) (Fig. 2b).We also counted the proportion of OPCs or OLs in the corpus callosum of control and Atrx FoxG1Cre brain cryosections at P14, marked by PDGFRα and adenomatous polyposis coli (APC), respectively.While the proportion of PDGFRα + OPCs is lower in Atrx FoxG1Cre mice (12.8%) compared to controls (23.9%), it was not improved upon T4 treatment (11.7%) (n = 3 each genotype; p = 0.0018, one-way ANOVA) (Fig. 2c).The proportion of differentiated APC + OLs was decreased in the Atrx FoxG1Cre corpus callosum (11%) compared to controls (29.2%) and was partially recovered (20.4%) following T4 treatment (n = 3 for each genotype; p = 0.0018 one-way ANOVA) (Fig. 2d), suggesting that thyroxine can only remediate differentiation defects.
To confirm that T4 administration promotes the differentiation of ATRX-null OPCs, we performed qRT-PCR for several established myelin genes including Mag, Mog, Mbp and Plp1 (Proteolipid protein 1).The level of these RNA transcripts was elevated in T4-vs PBS-treated Atrx FoxG1Cre mice, but the extent of rescue did not reach control levels for Mag and Mog (n = 3-5 each genotype; Mag p = 0.0008; Mog p = 0.0001, Mbp p = 0.17, Plp p = 0.001 by one-way ANOVA) (Fig. 2e).Similarly, Western blot analysis of MAG, MOG and MBP showed some rescue of myelin protein levels in T4-treated Atrx FoxG1Cre forebrain extracts (n = 3 each genotype MAG p = 0.026; MOG p = 0.027, MBP p = 0.035 by one-way ANOVA) (Fig. 2f).Immunofluorescence staining of MOG and MBP in Atrx FoxG1Cre forebrain cryosections also showed improved myelination after T4 administration (Fig. 2g).Taken together, these results suggest that hypomyelination in Atrx FoxG1Cre mice is due to a reduced number of OPCs and OLs and that T4 treatment stimulates OPC differentiation without increasing OPC numbers, which could explain the incomplete effect of T4 treatment on the extent myelination.

Targeted ablation of Atrx in neurons does not affect myelination
The results of the T4 experiments suggested that ATRX is required in the central nervous system to support normal myelination, but it was not clear whether this effect is cell autonomous in OPCs or caused by ATRX loss in other cell types that provide trophic support to OPCs.To resolve whether ATRX regulates myelination intrinsically in OPCs or indirectly via neuronal signaling (or both), we generated mice that lack ATRX in forebrain excitatory neurons using the NexCre driver mice for Atrx loxP recombination 53 .We confirmed the absence of ATRX nuclear protein in cortical neurons by co-immunofluorescence staining of ATRX and the neuronal marker NeuN (Fig. S1a).We next examined myelin protein expression in the forebrain by immunofluorescence staining of P20 brain cryosections and no obvious difference in MBP and MOG protein was observed between control and Atrx NexCre mice (Fig. S1b).Furthermore, MAG, MOG and MBP protein levels were equivalent to controls when assessed by immunoblot of P20 Atrx NexCre forebrain extracts (n = 3 each genotype; Student's T-test, MAG p = 0.627; MOG p = 0.917, MBP p = 0.425) (Fig. S1c).These results establish that ATRX is not required in neurons of the developing brain for proper signaling to OPCs and for normal production of myelin.

Loss of ATRX in OPCs leads to hypomyelination
We first verified that ATRX is expressed in OPCs and OLs in the mouse brain by co-staining of ATRX with PDGFRα and APC, respectively (Fig. S2).Next, we targeted ATRX deletion in primitive OPCs (priOPCs) and OPCs using the tamoxifen-inducible Sox10CreER T2 mice, which upon mating with Atrx LoxP mice produce Atrx Sox10-iCreERT2 (Atrx Sox10Cre ) mice 54 .The Cre-sensitive nuclear membrane Sun1GFP reporter was also introduced to allow fate tracking of Cre-expressing cells (Fig. 3a) 55 .Cre recombination was induced by daily intraperitoneal (IP) injection of nursing dams for three successive days (pups at P1-P3) 56 and the mice were analyzed at P20 (Fig. 3a).Atrx depletion was confirmed by isolating nuclei from control and Atrx Sox10Cre P20 forebrains and sorting the Sun1GFP + OPC nuclei.qRT-PCR analysis confirmed a 74.2% decrease in Atrx transcript levels in Atrx Sox10Cre Sun1GFP + nuclei compared to control Sun1GFP + nuclei (n = 4 each genotype; Student's T-test, p = 0.0096) (Fig. 3a).
We next performed RNA-seq of Sun1GFP+ nuclei obtained from control and Atrx Sox10Cre forebrain and identified 3479 altered genes (aggregated transcripts, Lancaster method, p value < 0.05).Gene ontology analysis identified categories of enrichment for biological processes "myelination", "axon ensheathment" and "ensheathment of neurons", as well as "glial cell differentiation" and "actin polymerization and depolymerization".Moreover, the top cellular component category of enrichment was "myelin sheath" and many of the molecular function enrichment categories featured aspects of cytoskeletal biology, possibly reflecting extensive changes in cell shape and membrane architecture during myelination (Fig. 3b).Cluster analysis of only the downregulated genes further emphasized that numerous genes implicated in myelination, gliogenesis and cell morphogenesis involved in differentiation are downregulated in ATRX-null priOPCs and their progeny (Fig. 3c).
To verify potential defects in OPC differentiation and myelination, we performed immunofluorescence staining for APC, a marker of differentiated but immature OLs (iOLs) in P20 control and Atrx Sox10Cre cortical sections.There was a significant reduction in the number of Sun1GFP + ATRX-null cells that co-expressed APC (41%) compared to Sun1GFP + control cells (61.4%) (Fig. 3d) (n = 3 each genotype; p = 0.020 Student's T-test), indicating a lower number of differentiating iOLs.RNA transcripts of mature OLs (mOL)-specific genes Mag, Mog, Mbp were also significantly decreased in P35 Atrx Sox10Cre optic tract (n = 3-4 each genotype; Mag p = 0.10, Mog p = 0.017, Mbp p = 0.010 Student's T-test) (Fig. 3e).Furthermore, immunofluorescence staining for MOG and MBP in brain cryosections at P20 showed decreased level of these myelin proteins in the corpus callosum and cortex of Atrx Sox10Cre mice compared to controls (Fig. 3f).Reduced myelin protein expression in Atrx Sox10Cre mice was confirmed by western blot analysis of MAG, MOG and MBP (n = 4 each genotype; MAG p = 0.21, MOG p = 0.015, MBP p = 0.036 Student's T-test) (Fig. 3g).These results demonstrate that ATRX-null priOPCs/OPCs have a reduced capacity to differentiate into iOLs and mOLs, leading to reduced myelination.
Loss of Olig2 expression in ATRX-null cells was also observed in primary glial cell cultures.Cre-mediated recombination was induced by administration of tamoxifen to nursing dams for two days (age of pups P0-P1).P3 cortices of Atrx Sox10Cre or control mice that also express the Ai14-TdTomato reporter 59 were dissociated and cells grown in mixed glial co-culture in which OPCs are expanded on top of an astrocyte monolayer (Fig. 4e) 60,61 .Immunofluorescence staining revealed that many ATRX-null Ai14 + cells do not express Olig2 after 5, 6 and 9 days in vitro (DIV) (Fig. 4 f, g) and that these cells appear elongated and flattened instead of the typical rounded shape of OPCs (Fig. 4f, h).We also detected a significant increase in apoptosis of Ai14+ ATRX-null rounded OPCs compared to control cells at both DIV5 and DIV6 (Fig. 4i, j).Ki67 staining showed that the same proportion of rounded Ai14+ cells proliferate in the presence or absence of ATRX at DIV5.However, a significant decrease in Ai14 + /Ki16+ cells was detected at DIV6 (Fig. S3).Overall, these results demonstrate that a subset of ATRX-null OPCs lack Olig2 expression and either undergo apoptosis or change morphology.In addition, the proliferation of ATRX-null OPCs was not initially affected but was decreased by 6 DIV compared to control cells.

ATRX co-occupies the Olig2 locus with other OPC fate regulators and promotes an active chromatin state
We next performed ATRX chromatin immunoprecipitation on cultured primary mouse OPCs and identified 42,820 ATRX peaks using MACS2 62 , with the bulk of ATRX chromatin occupancy identified two highly represented DNA motifs corresponding to Oct6 (Pou3f1) and Brn1 (Pou3f3) (Fig. 5b) from ChIP-seq data previously done in NPCs 63 .These transcription factors are not known to contribute to OPC identify or differentiation and their expression may not be substantial in OPCs.Not surprisingly, when we compared ATRX enrichment +/−3 kb from Oct6 and Brn1 binding site midpoints in OPCs, we only observed weak ATRX enrichment flanking these transcription factor binding sites.Interestingly, ATRX binding was more evident at Sox2 peaks, which was analyzed in parallel, as a control (Fig. 5c).These results suggest that in OPCs, ATRX may be co-binding chromatin with a POU domain transcription factor that has yet to be identified.
We then turned to other established critical factors involved in OPC differentiation, including HDAC3 64 , CHD7 65 , CHD8 66,67 and OLIG2 3,58,68 , for which data was available for comparison.Comparing ChIP-seq of ATRX and these factors in OPCs showed substantial cooccupancy of ATRX with chromatin remodelers CHD7 and CHD8, but not with HDAC3 or OLIG2 (Fig. 5d, f).CHD7 and CHD8 are ATPdependent chromatin remodeling proteins involved in OL differentiation and myelination 65,66 .CHD8 has been implicated in the establishment of OPC identity, while both CHD7 and CHD8 target enhancers of key myelinogenic genes 65 , which are marked by histone H3 acetylated at lysine 27 (H3K27Ac) 69 .Comparison of H3K27Ac ChIPseq peaks from OPCs, immature OLs (iOLs) and mature OLs (mOLs) revealed elevated ATRX binding at active enhancers in these cell types, although it was more pronounced in OPCs compared to iOLs and mOLs (Fig. 5e, f).ATRX directly binds the Olig2 gene locus at regulatory sites defined by H3K27Ac and CHD8, but not for CHD7 (Fig. 5g).An overlap of ChIP-seq peaks between ATRX, H3K27Ac and CHD8 is also observed at numerous other genes involved in OL fate specification and differentiation, such as Nkx2-2, Sox8, Thra and Mbp (Fig. S4).Given the strong association between H3K27Ac and ATRX, we performed ChIP-qPCR of H3K27Ac in control and Atrx Sox10Cre optic nerves.The results demonstrate a significant reduction of this histone posttranslational modification at the Olig2 gene locus in the absence of ATRX, corresponding to reduced gene expression (Fig. 5h).
We further compared the list of differentially expressed genes identified by RNA-seq, to cell type-specific gene lists 70 and determined that the majority of downregulated genes are highly expressed not only in OLs, but also OPCs, suggesting that perhaps OPC determination is compromised in the absence of ATRX (Fig. 6a).In addition to Olig2, several other transcription factors involved in OPC fate 71 were downregulated according to RNA-seq data, including Olig1, Sox8, and Zfp36l1 (Fig. 6b).The latter codes for a transcription factor that promotes differentiation of multipotent progenitors into pri-OPCs 71 suggesting that ATRX-null cells fail to express early determinants of OPC and pri-OPC identify.Indeed, RT-qPCR analysis shows a reduction in the expression of the OPC markers Pdgfrα, Cspg4 (NG2) and Gpr17 (n = 3-4 each genotype; Student's T-test, Pdgfrα p = 0.017, Cspg4 p = 0.032, Gpr17 p = 0.0004) in the Atrx Sox10Cre mice as compared to controls (Fig. 6c).The priOPC-expressed genes Olig1 and Sox10 were similarly reduced at the transcript level but did not reach the significance threshold of p < 0.05 (Fig. 6c) (n = 3 each genotype; Olig1 p = 0.081, Sox10 p = 0.133 Student's T-test).Moreover, Sun1GFP+ ATRX-null OPCs in the corpus callosum stain less frequently for PDGFRα (24.9%) compared to controls (41.1%) (Fig. 6d, e  was also performed on mixed glial cultures to examine OPC development over time.We observed that a significantly decreased proportion of ATRX-null Ai14+ cells express PDGFRα at 6 and 9 DIV compared to control cells and that more Ai14+ cells lack the expression of both SOX10 and OLIG2 at 6 and 9 DIV in Atrx Sox10Cre compared to control mice (Fig. 6f-h).We conclude that ATRX ablation in postnatal Sox10expressing priOPCs and OPCs leads to a loss of progenitor identity.

Loss of ATRX leads to ectopic astrogliogenesis
Ablation of the transcription factor Olig2 in OPCs in the postnatal cortex has been reported to induce differentiation into astrocytes [5][6][7] .This observation, combined with the fact that many ATRX-null OPCs take on an atypical morphology reminiscent of astrocytes in vivo and in vitro, prompted us to investigate whether ATRX-null OPCs adopt a more malleable state conducive to an alternative fate choice.To answer this question, we first compared RNA-seq data to the list of the most enriched genes in astrocytes 70 , and found that many astrocyteenriched genes were upregulated in ATRX-null cells (Fig. 7a).As many of the genes in this list are merely enriched in, but not specific to astrocytes, we then performed immunofluorescence staining of known astrocyte markers in control and Atrx Sox10Cre brain cryosections at P20.We observed increased S100 calcium-binding protein β (S100β) staining in the corpus callosum and cortex in Atrx Sox10Cre mice (Fig. 7b).Cells co-expressing the endogenous Sun1GFP and S100β (by immunostaining) were counted, and the results show a 20% increase in the number of Sun1GFP + cells that co-express S100β in Atrx Sox10Cre compared to control corpus callosum (n = 3 each genotype; Student's Ttest, p = 0.003) (Fig. 7c).S100β can sometimes stain OLs 72 and indeed we observed that a proportion of control Sun1GFP + OPCs express S100β (34.2%).We thus used another well-known astrocyte marker, glial fibrillary acidic protein (GFAP).Like the results obtained for S100β, we observed increased GFAP staining in the cortex of Atrx Sox10Cre mice (Fig. 7d).Cell counts of GFAP + Sun1GFP + cells showed a 15.2% increase in the proportion of Sun1GFP + cells that co-stain with GFAP in Atrx Sox10Cre compared to control in the corpus callosum (n = 3 each genotype; Student's T-test, p = 0.0074) (Fig. 7e).
Utilizing the Ai14 reporter to assess Atrx Sox10Cre cell morphology, we could easily see that a subset of ATRX-null Ai14 + cells display a highly branched morphology typical of protoplasmic astrocytes as previously observed in Olig2 knockout mice 6,7 .These cells coexpressed GFAP (Fig. 7f) and the astrocyte specification factor NFIA 73,74 (Fig. 7g) indicating that a subset of ATRX-null cells gained the capacity to at least initiate astrocyte differentiation in vivo.We confirmed this finding in primary glial culture, where at 5 and 6 DIV, significantly more Atrx Sox10Cre derived cells (Ai14 + ) expressed GFAP compared to control cells (Fig. 7h).Moreover, we observed that more Ai14+ GFAP+ cells lack Olig2 expression in the absence of ATRX (Fig. 7i).These results suggest that a portion of ATRX-null cells initiate an astrocytic differentiation program, instead of the Olig2-driven oligodendrocyte lineage pathway.

Discussion
ATP-dependent chromatin remodelers such as Brg1, Brm, CHD7, Chd8 and Ep400 have emerged as critical factors in the regulation of the OL lineage progression and myelination (reviewed in Parras et al. 2020) 75 .Still, many questions remain as to the exact mechanisms by which they exert their effects.In the present study, we add ATRX to the list of chromatin remodelers involved in oligodendrogenesis and report on ATRX-mediated regulation of myelin development through both systemic and cell-autonomous mechanisms.The Atrx Foxg1 Cre mice exhibit CNS hypomyelination that can partially be rescued by early postnatal administration of thyroid hormone.Cell type-specific inactivation of ATRX in OPCs, but not in neurons, also resulted in hypomyelination, showing that ATRX loss can have systemic as well as cell-autonomous roles in developmental myelination.We determined that ATRX directly binds to and activates the Olig2 gene at regulatory sites and promotes an active chromatin state (H3K27Ac).Consequently, Olig2 protein level is reduced in a subset of ATRX-null OPCs, leading to a more permissive precursor state, perhaps through modified chromatin accessibility, allowing for ectopic astrogliogenesis (Fig. 7j).
We first demonstrated that myelination is defective in Atrx FoxG1Cre mice, which have low levels of circulating T4 45,46 .The thyroid hormone receptor is a transcriptional activator for several myelin genes, promoting differentiation of OPCs [23][24][25] .We observed a significant yet partial rescue in myelinogenic genes and proteins in Atrx FoxG1Cre mice following T4 supplementation.Whereas the number of APC-positive iOLs increased following T4 administration, the number of PDGFRαpositive OPCs was not restored to control levels.Furthermore, production of Olig2-expressing OPCs in Atrx FoxG1Cre mice was not improved after thyroid hormone treatment, indicating that ATRX may be essential for OPC determination, proliferation or survival involving a mechanism distinct from T4 regulation.
Neuronal communication with OLs is emerging as a key regulator of myelination.Neurons have been proposed to be necessary for adaptive myelination 76,77 and electrical activity from neurons has been shown to induce OPC proliferation and remyelination 78 .ATRX is absent in all cell types of the forebrain of Atrx FoxG1Cre mice, and it was thus conceivable that deletion of Atrx in neurons could cause a reduction of the OPC pool and subsequent myelination.Nonetheless, specific deletion of ATRX in neurons did not result in defects in the development of white matter.Conversely, deletion in Sox10-expressing pri-OPCs and OPCs had substantial deleterious effects on myelin production.
Our data indicate that in OPCs, ATRX binds several sites across the Olig2 gene locus and promotes gene expression partly by helping to promote H3K27Ac via unknown mechanisms.Olig2 is a key determinant of OPC and OL fate, by promoting a cascade of gene expression, but also by directly suppressing astrocytic fate in conjunction with Olig1 3 .Loss of Olig2 can explain the reduced expression of Pdgfrα, since it is required for induction of this gene in cooperation with Sox10 8,9 .Pdgfrα is essential for OPC proliferation and survival and the decreased expression of this gene might explain the reduced proliferation and increased cell death observed in Atrx Sox10Cre mixed glial cultures.Depletion of the OPC pool can in turn cause the reduction in myelinating OLs and hypomyelination observed in Atrx Sox10Cre mice.
The gliogenic switch observed upon loss of ATRX is most likely mediated by changes in the epigenetic and chromatin landscape.Two factors involved in this process are the histone deacetylase HDAC3 and the histone acetylase P300 64 .However, we found no evidence of overlap between the occupancy of ATRX and these two factors in OPCs.Rather, we discovered substantial enrichment of ATRX at H3K27Ac, CHD7 and CHD8 binding sites across the genome.Like ATRX, CHD7 and CHD8 are ATP-dependent chromatin-remodeling enzymes and they can bind to the enhancer regions and near transcription start sites of myelinogenic genes 65,67 .However, CHD8 expression appears to play a bigger role in OPCs than CHD7.Chromatin occupancy of CHD8 in OPCs is greater than that of CHD7 66 , and CHD8 acts upstream of the Brg1-containing BAF complex and Olig2, both of which promote CHD7 expression 67 .Moreover, CHD8 occupancy in OPCs corresponds to proximal enhancer regions marked by H3K27Ac, while this is not the case for CHD7.Based on this, we predict that ATRX co-binds chromatin sites with CHD8 (as opposed to CHD7) in OPCs, although the purpose of utilizing two chromatin remodelers at enhancers will need to be resolved in future studies.It is interesting to note that CHD8 mutations cause congenital anomalies, intellectual disability and autism accompanied and severe white matter abnormalities [79][80][81] , akin to ATR-X syndrome, suggesting that there might be mechanistic overlap in white matter pathogenesis caused by disruption of these chromatin remodelers.
The fate switch observed upon loss of ATRX is reminiscent of the effects of Olig2 deletion in the mouse postnatal cortex 6,7 .The timing of Olig2 ablation has been shown to reflect the number of OPCs that revert to an astrocytic fate.When Olig2 is deleted constitutively in OPCs beginning at E16.5 there is almost complete conversion of these cells to astrocytes 6 .However, deletion of Olig2 at P2 or P18 results in a 50% and 25% conversion to astrocytes, respectively 7,82 .From these results, we predict that an earlier inactivation of Atrx in OPCs might yield a greater conversion to the astrocyte lineage in vivo.Our in vitro system is designed for the mass production of OPCs and allows for more cell division than would normally be observed in vivo, especially considering that the proliferation capacity of OPCs declines with age 83 .Accordingly, Atrx Sox10Cre mixed glial cultures exhibit a more drastic increase in conversion to astrocytes over a period of 9DIV.Mechanistically, Olig2 has been reported to directly repress key astrocytic fate determinants, including NFIA and GFAP, both of which were inappropriately induced in ATRX-null OPCs.NFIA can convert primed chromatin to active chromatin during early stages of astrocyte differentiation 73 , and has been shown to activate Gfap gene expression 84 .Although inactivation of Olig2 in OPCs causes a fate switch to astrocytes, other studies have revealed a more complex role of Olig2 in glial progenitors and astrocytes depending on the targeted cells, timing and brain region.For example, Olig2 has been reported to be transiently expressed in immature astrocytes of postnatal subventricular zone and to be required for white matter astrocyte maturation (Marshall 2005, Cai 2007).
Overall, our study suggests that myelination deficits caused by ATRX deficiency stems from cell-intrinsic functions of ATRX in the oligodendrocyte lineage, in addition to systemic mechanisms.Our findings could have implications in the treatment of myelin deficits in patients with disruptive ATRX mutations.In addition to the potential benefits of thyroid hormone treatment, adjusting glial cell-fate imbalances and overcoming intrinsic defects in oligodendroglial cell maturation and the ensuing developmental dysmyelination could be important therapeutic targets to improve white matter defects in ATR-X syndrome patients.

Animal husbandry and genotyping
All procedures involving animals were conducted in accordance with the regulations of the Animals for Research Act of the Province of Ontario and approved by the University of Western Ontario Animal Care and Use Committee (AUPs 2017-048, 2021-049 and 2021-062).Mice were housed at a temperature of 20-26 °C and humidity between 40-60%, were exposed to 12-h light/12-h dark cycles and fed water and regular chow ad libitum.All mice were purchased from The Jackson Laboratory except for the Atrx loxP mice which were provided by D. Higgs and R. Gibbons.Mice were euthanized with C0 2 inhalation or decapitation for neonatal mice (<10 days of age).The Atrx loxP mice have been described previously 85 (MGI:3528480).Briefly, loxP sites flanking exon 18 of the Atrx gene allow for deletion of this exon, destabilization of the mRNA and absence of the full-length ATRX protein 85 .Mating Atrx loxP female mice to males expressing Cre recombinase under the control of the Foxg1 promoter (129(Cg)-Foxg1 tm1(cre)Skm /J, RRID:IMSR_JAX:004337) 86 yielded male progeny with ATRX deficiency in the forebrain and anterior pituitary (Atrx loxP ;FoxG1Cre RRID:MGI:3530074) called Atrx FoxG1Cre for simplicity.Mating Atrx loxP female mice to males expressing Cre recombinase under the control of the Nex gene promoter 53,87 (Neurod6 tm1(cre)Kan , MGI:2668659) produced male progeny with ATRX deficiency in forebrain glutamatergic neurons (Atrx loxP ;NexCre or Atrx NexCre for simplicity).Mating Atrx loxP female mice to males expressing Cre recombinase under the control of the inducible Sox10 promoter 54 (CBA;B6-Tg(Sox10-icre/ERT2)388Wdr, MGI:5634390, RRID:IMSR JAX:027651), produced male progeny with Atrx deficiency in OPCs (Atrx loxP ;Sox10Cre or Atrx Sox10Cre ) upon tamoxifen treatment.Two Cre-sensitive reporter lines were bred into the Atrx loxP mice for nuclei sorting and lineage tracing.The Sun1GFP allele (B6;129-Gt(ROSA)26Sor tm5(CAG-Sun1/sfGFP)Nat / J , M G I : 5 6 1 4 7 9 6 , R R I D : IMSR_JAX:021039) encodes the Sun1GFP fusion protein (located in the nuclear membrane) upon Cre-mediated recombination 55 and the Tomato-Ai14 (Ai14) allele (B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J, MGI:3809524, RRID:IMSR_JAX:007914) 59 expresses the tdTomato protein with red fluorescence in the cytoplasm and nucleus upon Cre recombination.Ear punch genomic DNA was used for PCR genotyping of the Atrx floxed or wildtype alleles using the primers 17 F, 18 R and neoR as described previously 85 .The FoxG1Cre, NexCre, Sox10Cre, Tomato-Ai14 and Sun1GFP genotyping primers are listed in Supplementary Table 1.This study focuses on ATRX, which is X-linked.Female carriers display highly skewed X chromosome inactivation toward the mutant allele and are usually phenotypically normal.Additionally, Atrx FoxG1Cre males are unable to mate to produce Atrx FoxG1Cre females.

Microarray analysis
Total forebrain RNA (10 μg) was isolated from three pairs of littermatematched Atrx FoxG1Cre and control P17 mice using the RNeasy Mini kit (Qiagen Cat# 74104).cRNA was generated and hybridized to an Affymetrix Mouse Genome 430 2.0 Array at the London Regional Genomics Center (London, Canada).Probe signal intensities were generated using GCOS1.4(Affymetrix Inc., Santa Clara, CA) using default values for the Statistical Expression algorithm parameters and a Target Signal Data are presented as mean values +/− SD.Scale bar, 400 μm.h Images of Ai14 (red) and ATRX (green, top panels) or GFAP (green, bottom panels) in mixed glial cultures at 6DIV.Quantification of Ai14 + GFAP + cells is shown below at DIV5 (p = 0.0006, Ctrl n = 16 images scored from cultures established from 3 animals, Atrx Sox10Cre n = 15 images scored for 3 animals), DIV6 (p = 0.0092, Ctrl n = 13 images scored for cultures established for 3 animals, Atrx Sox10Cre n = 17 images scored for 3 animals) and 9DIV (p = 0.2268, Ctrl n = 14 images scored for 3 animals, Atrx Sox10Cre n = 23 images scored for 4 animals).Data are presented as mean values +/− SD.Scale bar, 400 µm.i Co-staining of glial cultures with GFAP (green), Olig2 (white) and DAPI (blue) shows decreased Olig2 staining in GFAP + Ai14 + ATRX-null cells (white arrowheads), quantified at DIV6 (p = 0.0035, n = 8 animals) and 9 DIV (p = 0.0002, n = 7 animals).Data are presented as mean values +/− SEM.Scale bar, 100 µm.j Model of the effects of ATRX inactivation in Sox10 + pri-OPCs and OPCs in the mouse postnatal forebrain.Loss of Olig2 and Sox10 expression reverts ATRX-mull OPCs and pri-OPCs to multipotent intermediate glial progenitor cells (iGCs), and gain the ability to differentiate into astrocyte-like cells in vitro and in vivo.All data were analyzed by two-sided Student's T-test; asterisks indicate significance p < 0.05.Created with BioRender.com,licence number GI244FLSLH.Source data are provided as a Source Data file.
of 150 for all probe sets and a Normalization Value of 1. Gene level data was generated using the RNA preprocessor in GeneSpring GX 7.3.1 (Agilent Technologies Inc., Palo Alto, CA).Data were then transformed (measurements less than 0.01 set to 0.01), normalized per chip to the 50th percentile, and per gene to control samples.Probe sets representing Atrx transcripts were removed (10 sets).The remaining probe sets were filtered by fold change ≥1.5 between control and Atrx FoxG1Cre samples, and by confidence level of p < 0.05.Significantly overrepresented GO categories were determined using the Enrichr web platform [88][89][90] .

Mixed glial primary culture
Cre recombination in control and Atrx Sox10Cre pups was induced by IP injection of 2 mg tamoxifen to lactating mothers for two consecutive days (P0-P1) 56 .At P3, mouse cortices were dissected into MEM and tissue dissociated by pipetting with a P1000.Samples were then incubated at 37 °C with OPC papain solution (Worthington Cat# LS003124) for 20 min with constant inversion to prevent tissue aggregation.Papain was deactivated with serum-supplemented media for 10 min at room temperature followed by trituration with a flame polished Pasteur pipette.Samples were centrifuged at 300 g for 5 minutes and pellets resuspended in 1 mL DMEM supplemented with 10% FBS (Gibco Cat # 12483020), 1% Antibiotic-Antimycotic (Gibco Cat # 15240062) and 1% GlutaMax (Gibco Cat # 35050061).The cell suspension from each tube was added to a pre-equilibrated poly-L-lysine (1 mg/mL) coated 6 well plate in 1 mL of media or flasks with 5 mL of media.Cultures were incubated in the presence of 8.5% CO 2 in a tissue culture incubator for 3 h to allow cells to attach to the poly-L-lysine substrate followed by a full media change.A 2/3 volume media change was performed every three days 60,61 .

Fluorescence microscopy, imaging and cell counts
Immunofluorescence images were captured using an inverted microscope (DMI 6000b, Leica) outfitted with a digital camera (ORCA-ER, Hamamatsu).Openlab software (PerkinElmer Version 5.0, RRID:SCR_012158) was used for image capture.Image processing was performed using Volocity (PerkinElmer Demo Version 6.0.1, RRID:SCR_002668) and Adobe Photoshop.Cell counts of brain sections and cultured cells were done in a blinded and randomized manner in control or experimental samples using Volocity (Perki-nElmer Demo Version 6.0.1, RRID:SCR_002668).For brain sections, counts were performed in 2-10 sections per biological replicate and 3-4 biological replicates were counted for each genotype.

Quantitative reverse transcriptase-PCR (qRT-PCR)
cDNA was prepared using 100 ng RNA from forebrain (P14 Atrx FoxG1Cre ), FANS-purified GFP + OPC nuclei or from optic tracts (P20 Atrx Sox10Cre ) (RNeasy Mini kit; Qiagen Cat# 74104) and 1.5 μL of 100 ng/L of random hexamers (Integrated DNA Technologies Cat# 51-01-18-26) were diluted to a final volume of 12 μL with RNAse free water.Samples were heated at 65 °C for 10 min followed by addition of 4 µL first strand buffer (Thermo Fisher Scientific Cat# 18064014), 2 µL 100 mM DTT (Thermo Fisher Scientific Cat# 18064014), 0.8 μL 25 mM dNTPs, 0.5 μL RNaseOut (Thermo Fisher Scientific Cat# 10777019), 0.5 μL Super-Script II Reverse Transcriptase (Thermo Fisher Scientific Cat# 18064014) and 0.5 µL RNAse free water per sample.Samples were then incubated at 30 °C for 10 minutes and 42 °C for 45 min and stored at −20 °C.cDNA was amplified with iQ SYBR Green Master Mix (BioRad Cat# 1708884) using the standard curve Ct method of quantification.Experiments were performed on a Chromo-4 thermocycler (MJ Research/BioRad) and analyzed with Opticon Monitor 3 and GeneX (BioRad) software.Technical duplicates were completed for each sample.Conditions for amplification were as follows: 40 cycles of 95 °C for 10 s, 55-60 °C for 20 s, 72 °C for 30 s, and a final melting curve generated in increments of 0.5 °C per plate read.Primer sequences are listed in Supplementary Table 1.

RNA-seq analysis
RNA-seq libraries were prepared with 90 ng of RNA using VAHTSTM Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme NR603-01) following the manufacturer's instructions.Libraries were sequenced at Canada's Michael Smith Genome Sciences Centre (BC Cancer Research, Vancouver, BC, Canada) using the Illumina HiseqX (Illumina Inc., San Diego, CA).On average, 50 million paired-end reads (150 bp) were obtained for each library.Raw reads were pre-processed with Trimgalore version 0.5.0, a wrapper tool around the sequence grooming tool cutadapt 92 with the following quality trimming and filtering parameters ('-phred33 -length 36 -q 5 -stringency 1 -e 0.1').Each set of paired-end reads was mapped against the Mus musculus GRCm38.p6primary assembly downloaded from Ensembl 93 release 94 (https://useast.ensembl.org/Mus_musculus/Info/Annotation)using HISAT2 version 2.0.4 94 .SAMtools was then used to sort and convert SAM files.The read alignments and Mus musculus GRCm38 genome annotation were provided as input into StringTie v1.3.3 95 which returned gene and transcript abundances for each sample.We imported coverage and abundances for transcripts into R using the tximport 96 R/Bioconductor package and conducted differential analysis of transcript count data using the DESeq2 R/Bioconductor package 97 .We used the independent hypothesis weighting (IHW) method 98 to weight P values and adjust for multiple testing using the procedure of Benjamini Hochberg (BH) 99 , transcripts p-values were then aggregated using the Lancaster method 100 .To generate the heatmap, gene level counts from the Stringtie results were transformed using variance stabilizing transformation (VST) method in DESeq2 97 .Z scores were then plotted using the pheatmap R package.Gene Ontology analysis was performed using Protein Analysis Through Evolutionary Relationships (Panther), http:// www.pantherdb.org 101.Gene cluster analysis was performed using TOPPCluster 102 .

Chromatin immunoprecipitation (ChIP)
For ATRX ChIP, cortices from fifteen P0.5 pups were dissected and OPCs were cultured for 9 DIV in a mixed glial culture.OPCs were then physically separated from other cells by shaking at 220 rpm overnight in a tissue culture incubator at 5% CO 2 60,61   .OPCs were pooled, homogenized and passed through a 70 µm strainer (Fisher Cat#22363548).For H3K27Ac ChIP, 3 to 4 optic tracts were dissected from either control or Atrx Sox10Cre P20 mice.Tissue was minced and triturated in DMEM and filtered through a 70 µm strainer (Fisher Cat#22363548).
OPCs were first crosslinked with 0.002 M ethylene glycol bis (succinimidyl succinate) (EGS, Thermo Fisher Scientific Cat # 21565) at room temperature for 45 min with constant mixing 103 .Both OPC and optic tract samples were crosslinked with 1% formaldehyde at room temperature for 20 min (OPCs) or 10 min (optic tracts) with constant mixing.Samples were quenched in 0.125 M glycine for 2 min at room temperature and centrifuged at 700 g for 5 min.Pellets were washed twice with 10 mL cold PBS and stored at −80 °C.
Chromatin lysates (50 µg per IP for OPCs, 20ug per IP for optic tracts) were precleared with 100 uL (OPCs) or 20ul (optic tracts) protein G Dynabeads (Invitrogen Cat# 10009D) for 1 h at 4 °C.Precleared lysate and antibody-bound beads were combined and incubated at 4 °C overnight with rotation.The next day, beads were washed five times and eluted for 15 min at room temperature followed by a second elution for 20 minutes at 65 °C103 .Crosslinking was reversed by adding 5 M NaCl at 65 °C for 4 h.Samples were treated with 1 µL 10 mg/mL RNase A at 37 °C for 30 min then 1 µL 20 µg/mL proteinase K for 1 h at 45 °C.1/10th of precleared chromatin lysate was treated the same and used as input.

ChIP-seq analysis
ChIP-seq libraries were made with Accel-NGS 1 S Plus DNA library preparation kit (Swift Biosciences) following the manufacturer's instructions.Libraries were sequenced with Illumina NovaSeq 6000 and ~25 million paired-end reads (150 bp) were obtained from each library.The paired-end reads in Fastq format were trimmed with Trim Galore (v.0.6.6), a wrapper tool around Cutadapt 92 package, and were then aligned to the mouse genome (mm10) using Bowtie2(v.2.4.4) with the default parameters 104 .SAMtools (v.1.12)was then used to create and sort BAM files from the aligned reads recorded in SAM format 105 .The duplicated reads were marked with MarkDuplicates function from Picard (v.2.26.3).The mitochondrial DNA reads and blacklist regions of the genome were filtered out using Bedtools intersect (v.2.30.0).MACS2 (v.2.2.7.1) 62 broadPeak mode was used to call the peaks from the filtered BAM files with input control.R package csaw (v.1.26.0) 106 was used to count the reads in 300 bp non-overlapping windows.Background noise was estimated by counting reads in 2000bp bins.We selected 300 bp windows that have a signal higher than log2(3) above background.Windows were then merged if less than 100 bp apart but did not extend above 5 kb width.Rtracklayer (v.1.52.1) 107 package was then used to export the filtered windows in a BED format.FindMotifsGenome.pl and annotatePeaks.plfunctions from HOMER (v.4.10) 108 were used to find enriched DNA motifs in the peak lists and annotate the peaks in the genome, respectively.The ATRX ChIP Bigwig track was generated using Deeptools2 bamCompare (v.3.5.2) 109 with the parameter "-scaleFactorsMethod SES" (input control was used for normalization).plotHeatmap and plotProfile functions of Deeptools2 were also used to compare ATRX enrichment at peaks of publicly available ChIP-seq datasets.H3K27Ac and Olig2 ChIP data was downloaded from NCBI Gene Expression Omnibus (GSE42454).UCSC lift-Over tool was used to convert peak coordinates from rn4 to mm10 genome assembly.The raw fastq files of HDAC3 and p300 in OPCs were downloaded from NCBI GSE76412.The reads were aligned to rn4 genome assembly using Bowtie2 (v.2.4.4).SAMtools (v.1.12)was then used to generate and filter BAM files.Peaks were called as described for ATRX ChIP.UCSC liftOver was then used to convert peak coordinates from rn4 to mm10 genome assembly.Chd7 and Chd8 ChIP peaks in OPCs were downloaded from NCBI GSE116601.Brn1, Oct6, and Sox2 ChIP-seq data from mouse neural stem cell line NS5 was downloaded (in wig format) from NCBI GSE69859.Wig files were converted to bigwig files using wigToBigWig from rtracklayer (v.1.52.1).Bigwig files were then converted to bedGraph files using UCSC bigWigToBed-Graph tool.UCSC liftOver was then used to convert peak coordinates from mm8 to mm10 genome assembly.

Statistical analysis
Statistical analysis was performed using GraphPad Prism6 software (6.05;GraphPad Software Inc.) and results are expressed as the mean +/− SEM or SD.Two independent data sets were compared with the Student's T-test (unpaired, two-sided).Multiple independent data sets were compared with a one-way ANOVA with post-hoc Tukey's HSD.P values of 0.05 or less were considered to indicate significance.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | Defective myelination in the ATRX-null mouse forebrain.a Microarray analysis of P17 control and Atrx FoxG1Cre forebrain reveals a significant downregulation (P < 0.05) of genes related to myelination and oligodendrocyte morphology (n = 3 animals for each genotype).b List of genes corresponding to the "Abnormal Myelination" category, along with fold changes and p-values.c Manhattan plot of Allan Brain Atlas 10X scRNA gene set enrichment from the list of downregulated genes.Each line on the x-axis denotes a single gene set and the y-axis measures the −log 10 (p-value) for each gene set.d Immunofluorescence microscopy of P20 brain cryosections stained with anti-MOG (green) and anti-MBP (red) antibodies confirms decreased levels of these myelin proteins in the cortex (Ctx) and corpus callosum (CC) of the forebrain of Atrx FoxG1Cre mice compared to controls (Ctrl) (n = 3 animals for each genotype).Scale bar, 500 µm.e, f Western blot analysis of P20 forebrain protein extracts showing significantly decreased levels of MAG (p = 0.0006), MOG (p = 0.0286) and MBP (p = 0.0009) in Atrx FoxG1Cre compared to control mice (n = 4 animals for each genotype) after normalization to β-actin protein levels.Data are presented as mean values +/− SEM and asterisks indicate p < 0.05 (Two-sided Student's T-test).Source data are provided as a Source Data file.

Fig. 2 |
Fig. 2 | Postnatal thyroxine treatment partially replenishes the number of APC + OLs and the expression of myelin proteins in the ATRX-null forebrain but does not restore the number of Pdgfrα + OPCs. a Outline of experimental design.Each arrow represents a subcutaneous (SC) injection of 0.1 mg/kg T4 or PBS to Atrx FoxG1Cre and control pups.Analyses were then performed at P14.Quantification of immunostained P14 cryosections demonstrates that the reduced number of b Olig2+ (p = 0.0013) and c Pdgfrα+ cells (p = 0.0026) observed in the Atrx FoxG1Cre mice is not rescued by T4 treatment (Olig2+ p = 0.0064, Pdgfrα+ p = 0.0016; n = 3 animals for each genotype).d The number of APC+ cells are partially rescued upon T4 treatment (p = 0.0435; n = 3 animals for each genotype).e qRT-PCR shows that transcript levels of several myelin genes are decreased in the P14 Atrx FoxG1Cre forebrain and partially restored following T4 treatment (Mag Ctrl n = 4, Atrx FoxG1Cre + PBS n = 5, Atrx FoxG1Cre + T4 n = 4; Mog Ctrl n = 3, Atrx FoxG1Cre + PBS n = 5, Atrx FoxG1Cre + T4 n = 4; Mbp Ctrl n = 4, Atrx FoxG1Cre + PBS n = 5, Atrx FoxG1Cre + T4 n = 5; Plp Ctrl n = 4, Atrx FoxG1Cre + PBS n = 5, Atrx FoxG1Cre + T4 n = 5 animals for each genotype).f Western blot analysis of MAG, MOG and MBP shows decreased protein levels in P14 Atrx FoxG1Cre compared to control forebrain extracts (MAG p = 0.0247, MOG p = 0.0261, MBP p = 0.0307) and partial rescue upon T4 treatment (MAG p = 0.5838, MOG p = 0.6298, MBP p = 0.4642; n = 3 animals for each genotype), normalized to β-actin protein levels.g Immunofluorescence staining of P14 brain cryosections highlights the decreased expression of MOG (green) and MBP (red) protein levels in the Atrx FoxG1Cre cortex (Ctx) and corpus callosum (CC) that are ameliorated following T4 treatment (n = 3 animals for each genotype).Scale bar, 500 µm.Data are presented as mean values +/− SEM, and groups with different letters significantly differ from one another.All data were analyzed by one-way ANOVA with post-hoc Tukey HSD.Source data are provided as a Source Data file.

Fig. 3 |
Fig. 3 | Defective differentiation of ATRX-null OPCs causes hypomyelination.a Sun1GFP + nuclei were sorted and used for RNA-sequencing.RT-qPCR analysis of Sun1GFP + sorted nuclei demonstrates reduced Atrx expression (p = 0.0096 n = 4 animals for each genotype).b Gene Ontology pathway analysis of significantly altered genes identified by RNA-sequencing (n = 3 animals for each genotype).c Cluster analysis of downregulated genes in Atrx Sox10Cre OPC/OL compared to control nuclei are related to cell differentiation, gliogenesis and the myelin sheath.d Cell counts show significantly fewer Sun1GFP+ cells co-staining with the mature OL marker APC (red) in the P20 Atrx Sox10Cre corpus callosum compared to controls.Scale bar, 100 µm (p = 0.00196, n = 3 animals for each genotype).e RT-qPCR analysis shows reduced expression of Mag (p = 0.1028), Mog (p = 0.0174) and Mbp (p = 0.0100) in Atrx Sox10Cre P35 optic tract compared to controls (Mag Ctrl n = 3, Atrx Sox10Cre n = 4; Mog Ctrl n = 3, Atrx Sox10Cre n = 4; Mbp n = 4 animals for each genotype).f Immunofluorescence staining reveals that myelin proteins MOG (green) and MBP (red) are reduced in P20 Atrx Sox10Cre cortex (Ctx) and corpus callosum (CC) compared to controls (n = 3 animals for each genotype).Scale bar, 500 µm.g Western blot analysis of MAG (p = 0.2075), MOG (p = 0.0149) and MBP (p = 0.0357) and quantification (right) confirms decreased expression of these proteins in P20 Atrx Sox10Cre mouse forebrain protein extracts compared to controls after normalization to β-actin (n = 4 animals for each genotype).Data are presented as mean values +/− SEM.All data were analyzed by two-sided Student's T-test; asterisks indicate significance of p < 0.05.The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.Source data are provided as a Source Data file.

Fig. 4 |
Fig. 4 | Loss of Olig2 expression and morphological changes in a subset of ATRX-null OPCs. a Immunofluorescence microscopy of P20 Atrx Sox10Cre corpus callosum shows expression of the Sun1GFP fusion protein in the nuclear membrane of OPCs upon tamoxifen treatment (left).P20 Atrx Sox10Cre treated with tamoxifen also express Sun1GFP but lack expression of nuclear ATRX (right) (n = 3 animals for each genotype).Scale bar, 100 µm.b Fate mapping of ATRX-null Sun1GFP + OPCs reveals frequent loss of the Olig2 transcription factor (white arrowheads).Scale bar, 100 µm.c Quantification of Sun1GFP + OPCs that lack Olig2 staining (p = 0.0127 Ctrl n = 4, Atrx Sox10Cre n = 3 animals for each genotype).Data are presented as mean values +/− SEM.d qRT-PCR analysis demonstrates significantly decreased Olig2 transcripts in Sun1GFP + Atrx Sox10Cre sorted nuclei compared to Sun1GFP + control nuclei (p = 0.0359, Ctrl n = 4, Atrx Sox10Cre n = 3 animals for each genotype).e Experimental timeline of tamoxifen (Tam) injections to nursing mothers at P0 and P1 (black arrows).f Images of Ai14 control and Atrx Sox10Cre mixed glial culture stained for Olig2 (green) at 9DIV.Scale bar, 200 µm.g The proportion of Ai14 + Olig2 + cells is lower in ATRX-null cultures at DIV5 (p = 0.0282, n = 16 images scored for cultures established from 4 animals of each genotype), DIV6 (p = 0.0001, Ctrl n = 14 images for 4 animals, Atrx Sox10Cre n = 21 images for 4 animals) and DIV9 (p = 0.003, Ctrl n = 15 images scored for cultures established from 3 animals, Atrx Sox10Cre n = 18 images for 3 animals).Data are presented as mean values +/− SD. h The percent of Ai14 + Olig2 - ATRX-null OPCs exhibiting a flattened morphology increases over time in culture, reaching significance at DIV9 (p = 0.0001, Ctrl n = 15 images scored from cultures established for 3 animals, Atrx Sox10Cre n = 18 images for 3 animals).Data are presented as mean values +/− SD. i Images of Ai14 control and Atrx Sox10Cre mixed glial cultures at 6DIV stained for TUNEL (green).Scale bar, 200 µm.j Quantification of TUNEL staining shows increased cell death of round-shaped ATRX-null OPCs at DIV5 (p = 0.0001, Ctrl n = 12 images scored from cultures established from 3 animals, Atrx Sox10Cre n = 19 images scored for 4 animals) and DIV6 (p = 0.0001, Ctrl n = 12 images scored for 3 animals, Atrx Sox10Cre n = 16 images scored for 3 animals).Data are presented as mean values +/− SD.All graphs with two groups were analyzed by twosided Student's T-test; asterisks indicate p < 0.05.All graphs with four groups were analyzed by one-way ANOVA with post-hoc Tukey HSD.The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | ATRX occupancy in OPCs is enriched at active promoters and promotes histone H3K27Ac post-translational modification at the Olig2 locus.a ATRX ChIP-seq in mouse primary OPCs reveals frequent ATRX binding at intergenic sites and introns (n = 1 animal for each genotype).b DNA motif enrichment of sequence corresponding to ATRX binding significantly correlate with Oct6 (p = 1e-299) and Brn1 (p = 1e-103) transcription factor binding sites identified in NPCs.c ATRX genome wide occupancy at ChIP-seq peaks of Sox2, Brn1 and Oct6 in NPCs.d ATRX occupancy at ChIP-seq peaks of HDAC3, Olig2, CHD7, CHD8 and H3K27Ac in OPCs.e C -occurrence of H3K27Ac and ATRX in OPCs, iOLs and mOLs.f Heatmaps of ATRX occupancy at ChIP-seq peaks of H3K27Ac, CHD8, CHD7 and OLIG2 in OPCs.g IGV view of the Olig2 gene showing ATRX ChIP-seq peaks (red) and how they align with H3K27Ac, CHD8 and CHD7 (green).h ChIP-qPCR of H3K27Ac demonstrates reduced enrichment at several sites at the Olig2 gene in OPCs the absence of ATRX (A p = 0.0097, B p = 0.0479, F p = 0.0001, n = 4 animals for each genotype).Data are presented as mean values +/− SEM; asterisks indicate significance of p < 0.05 analyzed by one-way ANOVA with post-hoc Tukey HSD.Source data are provided as a Source Data file.

Fig. 7 |
Fig. 7 | A subset of ATRX-null OPCs acquire astrocyte-like features in vivo and in vitro.a Heatmap of astrocyte-enriched genes upregulated in ATRX-null cells, based on Cahoy et al, 2018 70 .b Immunostaining of S100β reveals increased levels in the P20 Atrx Sox10Cre corpus callosum (CC) and cortex (Ctx) compared to controls.Scale bar, 100 µm.c Number of Sun1GFP + OPCs that stain positive for S100β (p = 0.0034, n = 3 animals for each genotype).Data are presented as mean values +/− SEM.d Immunostaining of the astrocyte marker GFAP shows increased expression in the P20 Atrx Sox10Cre mouse corpus callosum (CC) and cortex (Ctx) compared to controls.Scale bar, 100 µm.e Quantification of the number of Sun1GFP + OPCs that stain positive for GFAP (p = 0.0074, Ctrl n = 4, Atrx Sox10Cre n = 3 animals of each genotype).Data are presented as mean values +/− SEM.f Overlap of the Cre-sensitive Ai14 + reporter (red) and the astrocyte marker GFAP (green) in the Atrx Sox10Cre mouse brain in vivo (white arrowheads).Scale bar, 100 µm.(f') Higher magnification of portion of the image in (f), showing GFAP staining in an Ai14 + cell with astrocyte-like morphology.Scale bar, 25 µm.The extent of overlap of GFAP in morphologically altered Ai14 + cells is quantified in the graph on the right (p = 0.0064, Ctrl n = 6, Atrx Sox10Cre n = 5).Data are presented as mean values +/− SEM.g Immunostaining of NFIA (green) reveals increased expression in Ai14 + ATRX-null cells.Data is graphed on the right (p = 0.0009), Ctrl n = 16 images scored from cultures established from 4 animals, Atrx Sox10Cre n = 19 images scored for 4 animals.